The long-term objective of this proposal is to better understand the molecular basis of Y. pestis differentiation from the closely related species Y. pseudotuberculosis (Yptb), particularly with respect to its ability to cause disease. This proposal addresses the hypothesis that differential gene expression in response to environmental conditions is a critical determinant that distinguishes the plague bacillus from Yptb in nature. The specific aims are to: Aim 1: compare and contrast, using 2D-gel electrophoresis, patterns of Y. pestis and Yptb protein expression in a variety of biologically relevant conditions; Aim 2: identify, using mass spectrometry (MS), a subset of Y. pestis proteins that are differentially expressed in response to the environment. This group will include proteins that are expressed specifically by Y. pestis, as well as some that are similarly regulated by both Y. pestis and Yptb. MS analyses will be done on: a) proteins isolated from 2D gels analyzed in Aim 1; b) differentially-tagged and fractionated whole cell protein lysates; Aim3: perform preliminary characterization of selected genes from Aim 2, using molecular and bioinformatics approaches to evaluate each gene's potential for future study. Yersinia pestis, the etiologic agent of plague, is a major biological threat agent and a Class A select agent. Identification of Y. pestis proteins that are expressed during biologically relevant conditions will provide new targets that can be exploited for plague detection, treatment, and prevention, as well as insights into the pathogenesis of this critically important pathogen. Characterization of Y. pestis-specific genes and their proteins in future studies will also identify potential mechanisms by which Y. pestis emerged as a new pathogen distinct from Yptb.